Proliferative Role of Kv11 Channels in Murine Arteries

Figures

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  Figure 1. Expression of ERG in mouse vascular smooth muscle. (A,B) Results of quantitative PCR experiments showing expression of ERG1, 2 and 3 (A) and full length ERG1a isoform (B) normalized to GAPDH expression in a range of vascular beds. Each column is the mean of 4 tissues ± s.e.m. Data are expressed using the 2−ΔCt formula (C) Fluorescent images of smooth muscle cells from different vessels labeled with Kv11.1 antibody (left column) and smooth muscle actin (SMA, middle column). Secondary antibodies only (no primary Abs) are shown at the bottom. Each image is representative of 3 separate dispersals. Scale bar = 5 μM.

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  Figure 2. Electrophysiological recordings in isolated vascular myocytes. Examples of K+ currents recorded from portal vein (PV, A) or carotid artery (CA, B) myocytes in external solutions containing either 5 mM K+ (i) or 140 mM K+ (ii). Voltage protocols shown as inserts. All scale bars represent 50 pA and 50 ms. Panels (C,D) show representative traces in control conditions (gray arrows) or in presence of 1 μM dofetilide (black arrows) on currents recorded at −100 mV in PV (C) and CA (D) following a step depolarization from −50 to +40 mV. All traces are representative of >5 cells from at least 3 animals.

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  Figure 3. Effect of ERG channel activators on membrane currents. Examples of K+ currents evoked by membrane depolarization to +40 mV from −60 mV in the absence and presence of 10 μM NS1643 recorded from a PV myocyte (A) and CA myocyte (B). Cells were repolarized to −100 mV after the test step to +40 mV. The mean increase in currents at +40 and −100 mV produced by two structurally different ERG channel activators (10 μM NS1643 and 3 μM PD118057) is shown in panels (C,D), respectively. Each panel is the mean of 4–7 cells. Black columns = PV and white columns = CA. Panel (E) shows the effect of 1 μM paxilline on NS1643-stimulated currents at +40 mV in mCA myocytes (n = 8). Data are expressed as mean ± s.e.m. *p < 0.05 according to Student's t-test.

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  Figure 4. Effect of ERG channel modulators on membrane potential in whole carotid arteries. (A) Mean membrane potential (Em) in carotid arteries in the absence and presence of ERG blockers (1 μM dofetilide) and activators (3 μM PD118057). The ATP-sensitive K+ channel activator levcromakalim (10 μM) was applied as a control. Each column is the mean of 4–8 arteries ± s.e.m. *p < 0.05, ***p < 0.001 according to one-way ANOVA. (B) Lack of effect of 1 μM dofetilide on contractions induced by 10 μM phenylephrine in mouse aorta or carotid artery (mean ± s.e.m, n = 6). Statistical analysis has been performed using Student's t-test (p > 0.05, non significant).

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  Figure 5. Changes in the expression pattern of ERG channels in proliferating arterial smooth muscle. (A) Differences in expression of ERG1 in cultured femoral VSMCs were calculated using 18S as housekeeping gene and the contractile VSMCs expression levels as calibrator, using the 2−ΔΔCt formula, where ΔΔCt = ΔCt proliferative −ΔCt contractile and 2−ΔΔCt represents the expression fold change. Each bar is the mean of 6–10 data from 3 to 5 assays in duplicate for each condition. *p < 0.05; **p < 0.01; ***p < 0.001, according to Student's t-test. (B) Proliferation rate (measured as % of cells incorporating EdU) of cultured femoral VSMC in the absence of FBS, or in culture media supplemented with 5% FBS in the presence of vehicle (control condition) or the indicated drug combinations. **p < 0.01, ***p < 0.001 compared to control (vehicle), according to one-way ANOVA.